High throughput screening is a term used to describe a method where many discrete assays are performed in parallel. Currently, the most widely established techniques utilize 96-well microtiter plates. Such microtiter plates have been used for decades in research and clinical laboratory practice, as they provide an easy method for increasing assay throughput. Typically made of polystyrene, such microtiter or microwell plates provide 6 to 96 individual wells, in which a matrix of samples can be mixed with reagents, agitated, incubated and the like, either manually or with automated handling equipment. A particular advantage of microtiter plates is that, not only can numerous samples be quickly simultaneously assayed, but alternatively, a number of different assay conditions can be employed. To obtain quantitative assay results, a beam of light is commonly scanned into each well to obtain spectroscopic information from each well.
Assays can also be performed on a surface of each individual well, such as in a sandwich immunoassay. A variety of surface-modified polystyrene microtiter plates are commercially available from different suppliers designed to fit various applications, such as ELISA or cell culture. A variety of automated equipment is also commercially available to process microtiter plates and samples.
Their ability to increase assay throughput has made microtiter plates the choice for use in many assays, e.g. those involving selective or enhanced immobilization on the surface of the wells of such plates. U.S. Pat. No. 5,741,638 describes a process of immobilizing a particular oligonucleotide sequence in a microtiter well for specific high sensitivity detection. U.S. Pat. No. 5,610,287 describes a method for non-covalently immobilizing synthetic nucleic acid molecules upon the surface of a polystyrene support, such as a microtiter plate, to allow hybridization and other nucleic acid assays to be performed in a rapid and cost-effective manner. U.S. Pat. Nos. 5,667,976, 5,712,383, and 5,747,244 describe compositions and methods for covalently immobilizing nucleic acids onto appropriate surfaces, such as wells of a microtiter plate, for similar assay purposes. U.S. Pat. No. 6,180,769 describes a method for linking negatively charged macromolecules, such as DNA and RNA, to the plastic of a microtiter plate for assay purposes.
Ways for increasing the ability to wash and to exchange fluids within a well of a microtiter plate have also been described through the use of pervious materials, e.g. a porous structure, for the bottom of a microwell plates, examples of such are shown in U.S. Pat. Nos. 4,493,815, 5,326,533, 5,679,310 and 6,146,854. The use of a vacuum manifold beneath such a microtiter plate is alleged to allow controlled and rapid evacuation of fluid from such wells. U.S. Pat. No. 5,106,496 includes a circular membrane in the bottom of each well. Millipore Corporation sells 96-well plates having a membrane bottom as their MultiScreen™ plates.
Reducing the volume of each well in a microtiter plate may allow for higher well density in a microtiter plate and thus very high throughput analysis, perhaps even approaching that of biochips. U.S. Pat. No. 6,027,695 describes a device incorporating microwells of only 5 microliters each and suggests that microtiter plates may conceivably incorporate as many as 9600 wells. Some commercial plates presently offer 1536 wells in plates where the wells have working volumes of about 1 μl–10 μl. An example of the fluid handling and control that might be used with such microwell analysis systems is described in U.S. Pat. No. 6,225,061. U.S. Pat. No. 6,235,520 describes the use of substrates with high-density microwells for measuring the response of cells in each well for drug screening, and U.S. Pat. No. 4,734,192 shows the use of a separate membrane sheet.
More recently, plates with higher densities than 96 wells per microtiter plate, e.g. 384 and greater, have begun to be commercially developed in order to provide increased throughput and reduced reagent requirements. However, such higher well densities in a conventional microtiter plate assay present three main challenges: 1) the additional cost associated with producing such high density microtiter plates, 2) the difficulties with fluid-handling due to small well volumes, and 3) difficulties with optically obtaining accurate quantitative assay results. Accordingly, the search continues for solutions to these challenges, particularly to obtaining quantitative accuracy.